1. INTRODUCTION:

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are intermediate products of the cellular processes, exerting profound influences on both physiological functions and pathological states¹.

ROS are oxygen-centered free radicals that include superoxide anion (O2•-) hydroxyl radical (•OH), hydrogen peroxide peroxyl radical (ROO•), and alkoxyl radicals (RO•). Nitric oxide (NO), is known as primary RNS, produced via an enzymatic reaction catalysed by nitric oxide synthase (NOS) during the conversion of arginine to citrulline². Understanding the roles of these reactive species is crucial, given their implication in diverse biological mechanisms and diseases. ROS and RNS are pivotal in cellular signalling pathways regulating fundamental processes such as cell proliferation and apoptosis^3,4. The unregulated increase of ROS results in oxidative stress, defined by a disproportionate balance between the generation of reactive species and the cellular antioxidant defence systems, and this condition contributes to the various diseases^5,6. ROS and RNS are implicated in the damage of cellular macromolecules, including DNA, protein, and lipids and also contribute to chronic diseases⁷ while both species play a crucial role in the immune response, facilitating the elimination of pathogens⁸. However, aberrant production can instigate chronic inflammation and tumor development⁹.Antioxidants help counter ROS by preventing their generation¹⁰.Several synthetic antioxidants have been developed to mitigate the deleterious effects of ROS and RNS, but these compounds have demonstrated toxic effects during clinical trials. In contrast, natural bioactive compounds exhibiting antioxidant properties derived from medicinal plants, offer promising, safe, and cost-effective alternatives. Numerous studies have substantiated that medicinal plants represent an abundant natural antioxidant, comprising polyphenolic compounds, flavonoids, anthocyanins, and carotenoids^11,12,13. The antioxidant efficacy of bioactive compounds such as polyphenols is highly dependent on chemical structure, including the position and number of OH groups, polarity, and bond dissociation energy required to remove a hydrogen atom^14,15, whereas flavonoids can directly scavenge oxygen free radicals and activate antioxidant enzymes¹⁶. Despite previous investigations on the antioxidant properties of numerous plant species, there exists a significant deficiency in the thorough understanding of Convolvulus pluricaulis, particularly its scavenging activity against hydroxyl radicals. Therefore, this work focuses on the quantitative phytochemical analysis and antioxidant activity of C. pluricaulis, a medicinal plant from Convolvulaceae family and exhibits a wide range of biological properties¹⁷. A single antioxidant assay may not adequately characterize the capability of plant extracts to scavenge different types of ROS and RNS. Therefore, it is imperative to employ several methods for assessing the scavenging activity of plant extracts against various forms of ROS^18,19. The current study investigates the antioxidant properties of C. pluricaulis through various in vitro assays, including an examination of its hydroxyl radical scavenging activity, which is reported here for the first time. Additionally, this research includes a comparative analysis of the results with previously published data, contributing significant insights for future explorations in this field.

2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

Trolox, phenazine methosulphate (PMS), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ABTS (2,2-azino-bis (3-ethylbenzothiazolin-6-sulfonic acid), catechin, and 2-thiobarbituric acid (TBA), nitroblue tetrazolium (NBT), were purchased from Sigma-Aldrich. Nicotinamide adenine dinucleotide (NADH), N-(1-naphthyl) ethylenediamine dihydrochloride (NEDA), trichloroacetic acid (TCA), Potassium ferricyanide, sodium nitroprusside (SNP), ascorbic acid, gallic acid, Folin–Ciocalteu were purchased from SRL Pvt. Ltd.(India). Ferric chloride (FeCl₃), sodium nitrite, vanillin, HCl, hydrogen peroxide and all other solvents and reagents used were of analytical-grade.

2.2 Plant material:

Convolvulus pluricaulis (whole plant) powder was obtained from Herbal Hills, Isha Agro Developers Pvt. Ltd, located at 36A/55AB in Lonavala Co.op. Indl.Est. Ltd., village-Nangargoon, Lonavala, Pune, Maharashtra, India. This fine powder is derived from the herb Convolvulus pluricaulis (Shankhapushpi), commonly used in Ayurvedic medicine for its cognitive enhancing properties and neuroprotective effects.

2.3 Extraction of polyphenols:

Polyphenols were extracted from Convolvulus pluricaulis through soxhlet extraction method. Briefly, 5 grams of finely powdered whole plant material was subjected to reflux with 100ml of methanol at 60°C for 2 hours. The filtrate was collected and the residue was then further extracted with 50ml of methanol for 1 hour at 60℃. After that, Whatman No. 1 filter paper was used to filter both filtrates and evaporated using rotary evaporator until dry. The obtained dried residue was dissolved in 20ml of methanol and stored at 8℃. This methanolic extract of C. pluricaulis (CPME) was used for further experiments.

3. QUANTIFICATION OF PHYTOCHEMICALS:

3.1.Total phenolic content (TPC)

Folin-Ciocalteu method was used to determine the TPC as described in the previously reported method²⁰. During experiment, 0.1ml of CPME was mixed with following reagents, 0.5ml of Folin-Ciocalteu reagent (1:2 dilution with distilled water), 2.8ml distilled water, and 2ml sodium carbonate solution (20% w/v) and allowed to stand for one minute at room temperature, resulting in the development of a blue colour, which was measured at 650nm with gallic acid as the reference standard. The total phenolic content (TPC) was quantified in milligrams of gallic acid equivalents per 100grams of the dry mass of Convolvulus pluricaulis powder (mg GAE/100g).

3.2. Total flavonoid content (TFC):

The TFC of CPME was determined using the aluminum chloride (AlCl₃) colorimetric assay, as previously reported²¹.In test tube 0.3ml sodium nitrite solution (5%) combined with 0.1ml of CPME. After 5 minutes 0.3ml of 10% aluminum chloride solution, 2ml of a 1M sodium hydroxide solution, and 2.4ml distilled water were then added, followed by measurement of absorbance at 510nm. Catechin served as standard and TFC was expressed as milligrams of catechin equivalent per 100g of the dry weight (mg CE/100g).

3.3. Total tannin content (TTC):

The TTC of CPME was quantified using a previously described method²². After adding 0.1ml of CPME to the 3ml of 4% vanillin prepared in methanol, 1.5ml of 8% HCl was added. The resultant mixture was maintained at room temperature for 20 minutes, and the absorbance was measured at 500nm and the catechin was used as the standard. TTC was presented as mg CE/100g.

4. ANTIOXIDANT ACTIVITIES:

4.1 DPPH free radical scavenging assay:

In this assay a synthetic DPPH free radical was used to assess the potential of plant extracts to scavenge or neutralize these radicals. In an alcoholic medium, DPPH displays a purple color, which changes into pale yellow upon the addition of the plant extract, indicating the reaction between the free radical and antioxidant molecules present in the extract under investigation. This assay was performed according to the previously described method²³. For the kinetic study, 1.6ml CPME (25 to 125µg/ml) was added to 2.4ml of DPPH (0.1mM) and absorbance was measured continuously at 517nm for 10 minutes with measurements taken every minute. A decrease in absorbance with time indicated the extent of scavenging activity exhibited by the CPME.

4.2 ABTS radical scavenging assay:

This assay is an ABTS^•+ radical cation-based assay used to check the antioxidant activity of plant extracts. In this study, the ABTS/potassium persulfate (PP) method was used, as reported by previous study^24,25. ABTS^•+ radical cation was generated by combining 179µl of PP (140 mM) in 10ml of ABTS solution (7 mM), as well as the combination was incubated in darkness for 16 hours. The absorbance of the produced ABTS^•+ radical cation solution had been adjusted to 0.700 at 734nm after dilution with ethanol. Five hundred microliters of CPME (5 to 25µg/ml) was mixed with 3ml ABTS^•+ solution, and the absorbance readings were recorded every 30 seconds for a duration of 10 minutes at 734nm. Various quantities of trolox (1 to 5µM) were added to the ABTS^•+ solution and absorbance was recorded at 734nm. Results were presented by Trolox-equivalent antioxidant capacity (TEAC).

4.3 Superoxide anion scavenging assay:

The superoxide anion scavenging capacity of CPME was evaluated using the previously reported method with slight modifications²⁶. Tris HCl (16mM, pH=8) was used to prepare the 60µM PMS, 468µM NADH, and 156µM NBT. Briefly, 1ml of NBT, 1ml of NADH, and 1ml of CPME (25 to 225µg/ml) were mixed and then 1ml of PMS (60µM) was added to start the reaction, and was incubated for 5 minutes and the absorbance was determined at 560nm using catechin as standard.

4.4 Hydrogen peroxide scavenging assay:

This assay was performed using the previously reported method²⁷ to determine H₂O₂ scavenging potential of CPME. H₂O₂ solution (40mM) was prepared in phosphate buffer (50mM, pH 7.4). Briefly, 1ml of CPME (10 to 50µg/ml),1.4ml phosphate buffer, and 0.6 ml of H₂O₂ solution were mixed. The reaction mixture was kept for 15 minutes and determined the absorbance at 230nm using catechin as the standard.

4.5 Hydroxyl radical scavenging assay:

The assay was performed by deoxyribose degradation method, following the previously reported²⁸ by two methods, (1) Non-site-specific assay: The reaction mixture containing 0.2ml of each reagent, such as 2-deoxyribose (10mM), FeCl₃ (10mM), ascorbic acid (1 mM), EDTA (1mM), hydrogen peroxide (10mM), and 1 ml of CPME (5 to 25µg/m) in sodium phosphate buffer (50mM, pH=7.4) and incubated for 1 hour at 37°C. Following incubation, 1ml of TBA (0.5%) and 1ml of TCA (10%) were added, heated for 30 minutes at 80°C, and determined the absorbance at 532nm. (2) The site-specific assay is similar to the non-site-specific version, except that EDTA was excluded from the chemical reagents. Catechin was used as standard.

4.6 Nitric oxide scavenging assay:

The nitric oxide (NO•) scavenging potential of CPME was performed by the previously reported method with some modifications²⁹. Prior to the experiment sodium nitroprusside (SNP) solution (15mM) was freshly prepared in phosphate buffer (0.2M pH 7.4). To the 2 ml of freshly prepared SNP solution, 1ml of CPME (10 to 50µg/ml) was added and kept for 150 minutes at room temperature. Following incubation, 2ml of Griess reagent, prepared using (2% H₃P0₄, 0.1% NEDA, and 1% sulfanilamide) was added and measured absorbance at 550nm . Catechin was used as the standard.

4.7 Reducing power assay:

The reducing properties of CPME was determined using previously reported method³⁰. A volume of 2 ml of CPME (10 to 50µg/ml) was mixed with 2.5ml of phosphate buffer (0.2M, pH 6.6) and 2.5ml of potassium ferricyanide solution (1%). Using the water bath, the resultant mixture was incubated at 50°C for 20 minutes. Following the period of incubation, 2.5ml of TCA (10%), 2ml of distilled water, and 0.5ml of ferric chloride (FeCl₃) solution (0.1%) were added. The absorbance of resulting solution was determined at 700 nm. A higher absorbance indicates stronger reduction potential.

Calculation of Percentage of Inhibition:

The percentage of inhibition for all assays, except the reducing power assay was determined using the following formula:

% Inhibition = [(Ac – A_S)/Ac] × 100

Where Ac = absorbance of the control, A_S = absorbance of the sample

5. STATISTICAL ANALYSIS:

The experimental results were analysed using GraphPad Prism, version 8, a statistical analysis program. To find statistically significant differences between groups, two-way analysis of variance (ANOVA) was employed. A significance level of p<0.05 is regarded as statistically significant.

6. RESULTS AND DISCUSSION:

6.1 Quantitative phytochemical analysis:

The quantitative phytochemical analysis of CPME was performed to assess TPC, TFC, and TTC, summarized in Table 1. Notably, the observed TPC value of CPME was significantly higher than the previously reported (7.364 ±0.4133mg GAE/g and 21.57±0.064mg GAE/g extract) by M.H. Shalavadi(2018)³¹ in chloroform and ethanol extracts of C. pluricaulis whole plant, respectively and TFC value was found to be higher than previously reported by M.H. Shalavadi (2018)³¹ in chloroform and ethanol extract reported, which were 103.2±10.6 and 227±5.6mg QE/g of extract, respectively. This study also reports the quantification of the total tannin content for the first time in CPME (Table 1) and was found to be 373.33±12.49mg CE/100g dry powder or 51.74±1.38 mg CE/ g of extract.

Table 1. Quantitative phytochemical analysis of methanolic extract of C. pluricaulis (CPME)

S. No	Phytochemical analysis	Results
1	Total phenolic content	574.66±13.93mg GAE/ 100g or 79.59±1.58mg GAE/g of extract
2	Total flavonoid content	462.66±41.43mg CE/100 g or 64.22 ±5.15mg CE/g of extract
3	Total tannin content	373.33±12.49mg CE/ 100 g or 51.74 ±1.38 mg CE/ g of extract

6.2 DPPH radical scavenging assay:

In this assay, the DPPH radical scavenging activity of CPME was evaluated by monitoring the decrease in absorbance after the addition of CPME to the DPPH radical solution. This study represents a kinetic evaluation of CPME against the DPPH radical, which was initially assessed in the absence of CPME and the standard ascorbic acid, revealing the constant absorbance over time (Figure 1.A). The addition of CPME and ascorbic acid to the DPPH radical solution resulted in a gradual decrease in absorbance with time. (Figure 1.B). In this study, CPME showed 87.69% scavenging activity at 125µg/ml with an IC₅₀ value of 39.92µg/ml. In contrast, Das et al., (2019)³² reported an IC₅₀ value of 434.78µg/ml for the hydroethanolic leaf extract, while Tripathi (2018)³³ observed 61.2% scavenging activity at 100µg/ml with an IC₅₀value of 86.62µg/ml in the hydro-methanolic leaf extract of C. pluricaulis. These findings indicate that CPME possesses effective DPPH free radical scavenging activity.

Figure 1A. Kinetic study of DPPH radical scavenging assay: (A) CPME and (B) Ascorbic acid.

Figure 1 B. Percentage of DPPH radical scavenging activity of CPME and ascorbic acid

6.3 ABTS radical scavenging assay:

This assay was employed to evaluate the ability of CPME to counteract the ABTS^•+ radical cation. This method demonstrated a significant reduction in the characteristic blue-green colour of the ABTS radical solution, which indicates the antioxidant activity of CPME as the ABTS^•+ radical cation is converted to its stable form³⁴. In the kinetic study, a non-linear nature of absorbance versus time was observed for different concentrations of CPME and ascorbic acid (Figure 2.A). Tripathi (2018)³³ reported ABTS^•+radical cation scavenging activity of hydro-methanolic leaf extract of C. pluricaulis (52.87% at 100µg/ml) with an IC₅₀value of 99.75µg/ml and was found to be lower than present study (82.39% at 25µg/ml) with an IC₅₀ value of 6.67 µg/ml, indicating superior ABTS^•+ radical cation scavenging activity of CPME (Figure 2B). The trolox equivalent antioxidant capacity (TEAC) value is a measure of the antioxidant capacity of samples, expressed in terms of the equivalent activity of trolox, a water-soluble vitamin E analogue. A higher TEAC value indicates greater antioxidant potential. In the present study, the TEAC value for CPME was determined to be 904±0.016µM/100g indicating potent efficacy of CPME as a ROS scavenger.

Figure 2 A. Kinetic study of ABTS radical scavenging assay: (A) CPME and (B) Ascorbic acid

Figure 2 B. ABTS radical scavenging activity of CPME and ascorbic acid

6.4 Superoxide anion scavenging assay:

The superoxide anion radical scavenging activity of CPME was studied using the PMS/NADH system. Superoxide anion radical was generated from NADH oxidation and assayed by reduction of NBT into formazan³⁵. Although superoxide anion is a weak oxidant and can produce harmful free radicals such as hydroxyl radical and singlet oxygen, thereby increasing oxidative stress in biological systems^36,37. Polyphenolic compounds present in plant extract act as antioxidant molecules and scavenge superoxide anion free radicals³⁸. Therefore, it is essential to investigate the antioxidant activity of plant extracts to effectively scavenge superoxide anion and mitigate their detrimental effects. Das et al., (2019)³² reported superoxide anion radical scavenging activity of 88.07% at 800µg/ml with an IC₅₀ value of 132.18 µg/ml in the hydroethanolic leaf extract of C. pluricaulis found to be significant with the present study (73.47% at 225µg /ml) with an IC₅₀value of 133.75µg/ml (Figure 3).

Figure 3. Superoxide anion scavenging activity of CPME and catechin

6.5 Hydrogen peroxide scavenging assay:

Hydrogen peroxide, another ROS, has been the subject of extensive research over the years due to its ability to enter cells and transform into highly reactive hydroxyl radicals, which are toxic to cells³⁹. In the present study, CPME exhibited a 71.82% hydrogen peroxide scavenging activity at 50µg/ml, with an IC₅₀ value of 34.42µg/ml, highlighting the potential of CPME for hydrogen peroxide scavenging (Figure 4). Das et al., (2019)³² reported 90.12% of hydrogen peroxide scavenging activity at 500µg/ml with an IC₅₀value of 7.82µg/ml in a hydroethanolic leaf extract of C. pluricaulis.

Figure 4. Hydrogen peroxide scavenging activity of CPME and catechin

6.6 Hydroxyl radical scavenging assay:

The current study investigates the hydroxyl radical scavenging activity of CPME for the first time. Hydroxyl radical (•OH) is the most reactive and toxic of all known ROS. It has the capability to damage proteins and DNA^40,41. The •OH scavenging activity of plant extracts can be assessed through deoxyribose degradation assay. This assay employs the Fenton reaction involving Fe³⁺-ascorbate-EDTA-H₂O₂ to produce •OH radicals and assess the ability of the plant extract to scavenge these radicals⁴². In this study, the non-site-specific hydroxyl radical scavenging activity of CPME was observed 64.30% at 25µg/ml, with an IC₅₀ value of 3.033µg/ml (Figure 5 A). Conversely, in the site-specific assay, the hydroxyl radical scavenging activity of 77.51% at 25µg/ml was observed, with an IC₅₀ value of 19.37µg/ml. These findings indicate that CPME exhibits significant inhibitory activity against hydroxyl radicals, highlighting its potential as a source of antioxidant molecules (Figure 5 B).

Figure 5 A. Non-site-specific hydroxyl radical scavenging activity of CPME and catechin. B. Site-specific hydroxyl radical scavenging activity of CPME and catechin

6.7 Nitric oxide scavenging assay:

Nitric oxide (NO) is produced in biological systems via an enzymatic reaction catalysed by nitric oxide synthase (NOS). This reaction involves the breakdown of arginine into citrulline and NO in the presence of molecular oxygen and nicotinamide adenine dinucleotide phosphate (NADPH)⁴³. Exploring the antioxidant activity of plant extracts against nitric oxide free radicals can lead to significant advancements in therapeutic applications. In the current study CPME exhibited 58.89% of NO scavenging activity at 50µg/ml with an IC₅₀ value of 39µg/ml. This is comparable to previous study by Das et al., (2019)³² reported 58.94% scavenging activity at 1000µg/ml with an IC₅₀ value of 333.25µg/ml in the hydroethanolic leaf extract of C. pluricaulis indicating that CPME also possesses potent inhibitory activity against nitric oxide (Figure 6).

Figure 6. Nitric oxide scavenging activity of CPME and catechin

6.8 Reducing power assay:

Bioactive compounds having reduction potential may react with potassium ferricyanide (Fe³⁺) to form potassium ferrocyanide (Fe²⁺), after which it combines with ferric chloride to form a coloured ferric-ferrous complex⁴⁴. This is the basis for the reducing power test and evaluation of antioxidant activity. The extracts reducing power as well as antioxidant activity directly correlates with the intensity of the colour development. Figure.7 showed the significant reduction ability of CPME and catechin as the concentration increases. Present study concluded that the reducing power assay reinforced the importance of CPME as a hydrogen donor, which is vital for neutralizing free radicals

Figure 7. Reduction potential of CPME and catechin

7. CONCLUSION:

This study conducted a quantitative phytochemical analysis and evaluated the free radical scavenging capabilities of Convolvulus pluricaulis. The results indicate that the methanolic extract of C. pluricaulis (CPME) is a significant source of phytochemicals, contributing to the scavenging of free radicals. Notably, this study is the first to demonstrate the reducing potential and protective hydroxyl radical scavenging activity of CPME. The antioxidant assay revealed that CPME exhibits potent scavenging activity against various reactive oxygen species (ROS) and nitric oxide (NO). These findings were contrasted with data that had already been published, offering a comprehensive understanding of CPME's antioxidant characteristics and highlighting its potential as a source of natural antioxidants. Future studies should aim to explore the bioactive compounds present in C. pluricaulis for their effectiveness, in vivo efficacy using animal models to better understand their potential therapeutic properties in treating disorders related to oxidative stress.

8. CONFLICT OF INTEREST:

There is no conflict of interest within this article.

9. ACKNOWLEDGEMENTS:

The authors gratefully acknowledge the management of VIT, Vellore, for the opportunity to publish this work and for the financial support provided through the seed grant (VIT seed grant -2019 and RGEMS-SG20230137).

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Received on 07.05.2025 Revised on 13.09.2025

Accepted on 06.12.2025 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3265-3272.

DOI: 10.52711/0974-360X.2026.00465

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